LC ESI QOTOF METHOD DEVELOPMENT AND NEW INSTRUMENTATION EVALUATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们的实验室使用两台带有Waters Acuity LCS的应用生物系统API QSTAR(Tm)Pulsar I QqTOF质谱仪和一台Agilent CHIP-LC Q-TOF质谱仪，用于表征ESI和MALDI合作研究项目中获得的复杂生物样品。目前，我们的重点是建立用于蛋白质组、糖组和脂类组学应用的常规毛细管LC-IDA-MS/MS，以及提交给MS资源的各种生物样品的表征。针对典型的蛋白质消化、脂类和碳水化合物，IDA MS/MS自动化功能的使用正在不断优化，并生成了批处理参数文件，以便于实验室成员使用。处理脚本已经过优化，可以在用于数据分析的工作站上使用。使用较小直径的柱组件可显著提高灵敏度，从而能够分析低浓度或限量的样品。在优化LC系统之后，进行方法开发研究，以补充在MS资源内进行的当前研究。正在为糖组学、脂组学和基于蛋白质组学的研究开发方法，包括磷酸化分析。将Advion Triversa机器人添加到Q-Star系统中需要进行大量工作来寻找最佳条件，并与Advion和应用生物系统进行密切交互。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our laboratory uses two Applied Biosystems API QSTAR(tm) Pulsar i QqTOF mass spectrometers with Waters Acuity LCs and an Agilent chip-LC Q-TOF mass spectrometer for the characterization of complex biological samples obtained from collaborative research projects by ESI and MALDI. Currently, our focus is on establishing routine capillary LC-IDA-MS/MS for proteomics, glycomics and lipidomics applications, in addition to characterization of the large variety of biological samples that are submitted to the MS Resource. Use of automated IDA MS/MS functions are continuously being optimized for typical protein digests, lipids and carbohydrates, and batch parameter files have been generated for ease of use by the members within the laboratory. Processing scripts have been optimized for use on workstations for data analysis. The use of the smaller diameter column assemblies offers a significant increase in sensitivity, enabling samples of low concentrations or limited quantity to be analyzed. Optimization of the LC systems is followed by method development studies to complement the current research undertaken within the MS resource. Method development is being pursued for glycomics, lipidomics and proteomics-based studies, including phosphorylation analysis. Addition of the Advion TriVersa robot to the Q-Star system has necessitated much work to find optimum conditions, and close interaction with both Advion and Applied Biosystems.